The ultimate objective of the proposed research is to further characterize kinetically and chemically the facilitated transport of hexoses in animal cells and to elucidate the mechanism of its regulation. We have previously applied a rapid kinetic technique, which allows the determination of accurate time courses of changes in intracellular concentrations of isotopic substrates to transmembrane equilibrium, to study 3-O-methylglucose inward and outward equilibrium exchange and zero-trans influx and efflux in suspensions of cultured Novikoff rat hepatoma cells. The experimental data conformed well to the simple carrier model of Stein and his collaborators. The kinetic parameters were estimated by computer fitting appropriate integrated rate equations based on this model by the method of least squares to experiments data. The results indicated that the hexose carrier of Novikoff cells is indifferent with respect to direction (in/out symmetry), but that the loaded carrier moves faster than the unloaded carrier (loaded/unloaded asymmetry). Computer simulations indicate that such carrier is uniquely suited for a regulation of sugar transport in that a selective stimulation of movement of unloaded carried would result in a marked increase in zero-trans influx of sugar, particularly at higher sugar concentrations. The proposed research is mainly designed to test this hypothesis. We plan to determine whether a loaded/unloaded asymmetry is a property of the hexose transporter of other cell culture lines as well as of untransformed normal cells. We will determine the effect of growth stage, of insulin, serum, tumor virus transformation and D-glucose starvation on the kinetic properties of the transporter as well as of chemical modifications such as induced by oxidizing, reducing and sulfhydryl reactive agents. We will also determine the kinetic parameters for the transport of other hexoses and assess the effect of inhibitors (cytochalasin B and phloretin). Of particular interest in all these experiments is to determine whether loaded/unloaded asymmetry of the carrier is affected and whether such changes correspond to a postulated regulatory effect. We hope that the results lead to a better understanding of the molecular mechanism of carrier function and its regulation.